This supplemental application seeks to extend the work funded under R24 HL64387 "Engineered Cardiac Morphogenesis: Stem Cells and Scaffolds" which is focused on the development of engineered cardiac muscle tissue. A key aim of this parent award (alluded to in the title) is to study the interactions of stem cells and stem cell-derived parenchymal cells on both established and novel tissue engineering scaffolds. As proposed and funded, the parent grant limited these investigations to murine stem cells. However, recent advancements in our understanding of human embryonic stem (hES) cells, as well as recognition of key differences in cell behavior between ES cells from murine and human origin, suggest that it is crucial to extend now our investigations of ES cell-scaffold interactions to include human ES cell experiments. Cultures of human ES ceils (NIH code: WA07) will be expanded, differentiated, and fractionated to yield Hes cell-derived cardiomyocytes. Either human ES cells or the hES cell-derived cardiomyoctyes will be seeded on established and novel scaffolds including: collagen gels; microfiber arrays and fiberporous structures; and several porous polymeric scaffolds developed and in use under the parent award. We will evaluate cell toxicity and cell proliferation. We will assess proliferation and cellular organization within the scaffolds, e.g. expression of cardiac markers and the formation of intercalated disks. The proposed work is a direct and logical extension of our funded aims to evaluate these same parameters using murine ES cells seeded on scaffolds. We expect the results of the proposed work with human ES cells and hES cell-derived cardiomyocytes will greatly aid the search for cell sources suitable for cardiac tissue engineering, and will clarify parameters for the use of human ES cells in tissue engineering in general. The demonstrated pluripotentiality of human ES cells combined with success in tissue engineering strategies has tremendous potential to address the vastly limited supply of tissues and organs for therapeutic use, and thus success will be expected to impart enormous impact to the practice of regenerative medicine. [unreadable] [unreadable]